Female reproductive disorders, including precocious puberty, irregular menstrual cycles, and reduced fertility, are increasing among certain human populations. Endocrine-disrupting chemicals (EDCs) are common in the environment. Evidence from experimental animals, wildlife, and unintended human exposures suggests that EDCs may play a major role in causing these disorders. Epigenetic mechanisms (e.g., DNA methylation) may play a role as a link between environmental EDC exposures during development and the adult diseases. The epigenetic effects of exposure to EDCs (e.g., the pesticide methoxychlor, MXC) on other organs, including the uterus and mammary gland, have been studied. We have shown that developmental exposure to MXC alters gene expression in the ovary, leading to inhibition of follicular maturation and the ovulation, which are associated with gene-specific [(e.g., estrogen receptor (ER) 2] as well as global DNA methylation changes. The exposure causes reproductive dysfunction, such as early puberty, irregular cycles, reduced fertility, and premature aging. Preliminary studies also suggest that the MXC exposure cause morphological and gene expression alterations in postnatal day (PND) 7 ovary. However, the full extent of the epigenetic effects, how they are altered by age, the underlying developmental mechanisms, and the modulatory roles of common EDCs (e.g., bisphenol A (BPA) and genistein) on MXC-induced epigenetic alterations are not known. Our hypothesis is that developmental exposure to EDCs alters the epigenome (e.g., DNA methylation) and gene expression in the ovary in follicular stage-dependent and gonadotropic hormone-responsive manner, leading to reproductive dysfunction in adulthood. We will test this hypothesis with three specific aims: Aim 1 is to determine global age-dependent DNA methylation and gene expression changes in the ovary following developmental MXC exposure, using array- based approaches at PND7 and PND60. We will determine sequence-specific DNA methylation changes by bisulfite sequencing and how these changes may interfere with the transcription factor binding using chromatin immunoprecipitation assay. Aim 2 is to study the developmental mechanisms of MXC-induced DNA methylation and gene expression changes. We observed that ER2 expression was inhibited in pre- and early- antral follicles although the expression in earlier stages of follicles was not changed. Following isolation of different stage follicles, we will determine whether stage-specific follicular gene expression is due to stage- specific DNA methylation changes, and using a superovulation model, we will explore the role of gonadotropic hormones. Aim 3 is to determine the modulatory actions of BPA and genistein on MXC's effects. We observed that MXC, BPA, and/or genistein modulated each other's actions on early folliculogenesis in a stage-specific manner in vitro. To better understand the role of DNA methylation alterations in these modulatory actions among EDCs, we will study the effects of BPA and genistein on DNA methylation and gene expression changes induced by MXC in organ culture system in vitro as well as in whole animal in vivo. This approach will provide a broader understanding of the effects of EDCs and will help us develop a general theme for EDC- induced epigenetic alterations. These studies substantially improve our fundamental understanding of role of epigenetic mechanisms in gene expression and organ function, and how these mechanisms are influenced by environmental factors. A mechanistic understanding of epigenetic environmental disease etiology can lead to better prevention diagnosis, and therapies, thereby improving public health and reducing health care costs. PUBLIC HEALTH RELEVANCE: Exposure to environmental endocrine-disrupting chemicals (EDCs), such as the pesticide methoxychlor, during in utero or early postnatal developmental periods, may cause ovarian dysfunction and female infertility in adulthood via epigenetic mechanisms (e.g., DNA methylation). The objective of the proposed research is to identify the epigenetic mechanisms by which EDCs disrupt normal functioning of the ovary, leading to female infertility. A mechanistic understanding of the epigenetic effects of EDC exposure can lead to better prevention, diagnosis, and therapies, thereby improving public health and reducing health care costs.